Quick Reference

Bismark is run from the command line. Bowtie 2 or HISAT2 needs to be installed on your computer. For more information on how to run Bismark with Bowtie 2 please go to the end of this manual.

As of version 0.14.0 or higher, Bismark may be run using parallelisation for both the alignment and the methylation extraction step. Search for --parallel / --multicore for more details below.

First you need to download a reference genome and place it in a genome folder. Genomes can be obtained e.g. from the Ensembl or NCBI websites. For the example below you would need to download the Homo sapiens genome. Bismark supports reference genome sequence files in FastA format, allowed file extensions are either either .fa or .fasta. Both single-entry and multiple-entry FastA files are supported.

The following examples will use the file test_dataset.fastq which is available for download from the Bismark project or Github pages (it contains 10,000 reads in FastQ format, Phred33 qualities, 50 bp long reads, from a human directional BS-Seq library). An example report can be found in Appendix IV.

Command reference

Bismark ships as a single bismark binary. bismark <subcommand> is the recommended interface; the classic tool names remain fully supported as argv[0] aliases (a drop-in for existing pipelines and nf-core/methylseq).

Subcommand	Classic name	Purpose
bismark / bismark align	bismark	bisulfite read alignment
bismark dedup	deduplicate_bismark	deduplicate alignments
bismark extract	bismark_methylation_extractor	methylation extraction
bismark bedgraph	bismark2bedGraph	bedGraph + coverage from extractor output
bismark cov2cyt	coverage2cytosine	genome-wide cytosine report
bismark prepare	bismark_genome_preparation	build the bisulfite genome indices
bismark bam2nuc	bam2nuc	mono-/di-nucleotide coverage stats
bismark nome	NOMe_filtering	NOMe-seq GpC/CpG separation
bismark filter	filter_non_conversion	filter incompletely converted reads
bismark consistency	methylation_consistency	split reads by methylation concordance
bismark report	bismark2report	per-sample HTML report
bismark summary	bismark2summary	project-level HTML summary

Genome preparation

USAGE:

bismark prepare [options] <path_to_genome_folder>

A typical genome indexing could look like this:

bismark prepare --path_to_aligner /usr/bin/bowtie2/ --verbose /data/genomes/homo_sapiens/GRCh37/

Alignment

USAGE:

bismark [options] --genome <genome_folder> {-1 <mates1> -2 <mates2> | <singles>}

Typical alignment example:

bismark --genome /data/genomes/homo_sapiens/GRCh37/ test_dataset.fastq

This will produce two output files:

1. test_dataset_bismark_bt2.bam (contains all alignments plus methylation call strings)
2. test_dataset_bismark_SE_report.txt (contains alignment and methylation summary)

Note

In order to work properly the current working directory must contain the sequence files to be analysed.

Deduplication

bismark dedup --bam [options] <filenames>

This command will deduplicate the Bismark alignment BAM file and remove all reads but one which align to the the very same position and in the same orientation. This step is recommended for whole-genome bisulfite samples, but should not be used for reduced representation libraries such as RRBS, amplicon or target enrichment libraries.

Methylation extraction

USAGE:

bismark extract [options] <filenames>

A typical command to extract context-dependent (CpG/CHG/CHH) methylation could look like this:

bismark extract --gzip --bedGraph test_dataset_bismark_bt2.bam

This will produce three methytlation output files:

• CpG_context_test_dataset_bismark_bt2.txt.gz
• CHG_context_test_dataset_bismark_bt2.txt.gz
• CHH_context_test_dataset_bismark_bt2.txt.gz

as well as a bedGraph and a Bismark coverage file. For more on these files and their formats please see below.

Sample report

USAGE:

bismark report [options]

This command attempts to find Bismark alignment, deduplication and methylation extraction (splitting) reports as well as M-bias files to generate a graphical HTML report such as this example Bismark paired-end report for each sample in a directory.

Summary report

USAGE:

bismark summary [options]

This command scans the current working directory for different Bismark alignment, deduplication and methylation extraction (splitting) reports to produce a graphical summary HTML report, as well as a data table, for all files in a directory. Here is a sample Bismark Summary Report. The Bismark summary report is meant to give you a quick visual overview of the alignment statistics for a large number of samples (tens, hundreds or thousands of samples); if you only want to look at a single report please check out bismark report.